Fungi, and especially filamentous fungi, are widely used commercially because of their ability to secrete remarkably high levels of proteins
Among the filamentous fungi species belonging to the genus Aspergillus have a long history of commercial use for the production of endogenous and lately also heterologous proteins.
One disadvantage with most microorganisms used for the production of proteins is the inherent production of proteases which may subject a protein product of interest to degradation due to proteolysis.
Various ways of avoiding this have been envisaged. Among other solutions it has been suggested to delete or disrupt the genes encoding the various proteases. Unfortunately, the fungi produce a high number of proteases making such a solution more or less unrealistic.
A need is therefore persisting for strains of filamentous fungi exhibiting no or very low levels of protease production.
For a number of years it has been known that the regulatory gene areA which mediates nitrogen metabolite repression in A. nidulans influences the production of extracellular proteases (Arst & Cove, molec. gen. Genet. 126, (1973) 111-141).
The areA gene from A. nidulans has been cloned (Caddick et al., EMBO Journal 5, (1986) 1087-1090) and various modifications made to it to evaluate functions of different regions in the activator protein encoded by this gene (Stankovitch et al. Mol. Microbiol. 7, (1993) 81-87). Furthermore the gene coding the corresponding function in A. fumigatus apparently has been cloned recently (Hensel et al. 2nd European Conference on Fungal Genetics, Apr. 28 to May 1, 1994, Book of Abstracts, E11).
From the literature a single use is also known of a strain of A. nidulans of genotype argB areA1 as a host for the production of t-PA (Upshall et al. Biotechnology 5, (1987) 1301-1304). In this example only the argB genotype is used as a selection marker through its arginine prototrophy, while the areA genotype is simply a coincidence.
International Patent Publication No. WO 95/35385 discloses the deletion of the areA gene as a means for reducing the protease level in filamentous fungi.
Apart from the extracellular proteases, fungi also produce a number of intracellular proteases (also called endoplasmic).
Among these a serine protease of the subtilisin type produced by A. niger and designated PepC has been described, the gene expressing it cloned, and a deletion mutant described in EP 574 347 and in Frederick et al., Gene, 125 57-64 (1993)
A further such protease of the aspartic type designated PepE has been disclosed in Jarai et al., Gene, 145 171-178 (1994). the article discloses the cloning and characterisation of the pepE gene and speculates about the regulation of the pepE and pepC genes.
The present invention has as an object the alleviation of the need for protease free filamentous fungi.